All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.
Alfalfa (Medicago sativa L.) is an important forage species for hay and pasture, which has been referred to as the “Queen of the Forages” because of its high yields and feeding value. Alfalfa is recognized as the most widely adapted agronomic crop, as an effective source of biological nitrogen (N2) fixation, useful in the improvement of soil tilth, as an important source of protein yield/ha, and as an attractive source of nectar for honeybees. For a comprehensive review of the benefits of alfalfa as an agronomic crop, see Barnes et al., Highlights in the USA and Canada 1:2-24, In Alfalfa and Alfalfa Improvement, Hanson et al. (ed.), American Society of Agronomy, Monograph No. 29 (1988).
Although alfalfa originated in southwestern Asia, it is well adapted to a wide range of climates and soils in the United States. Alfalfa is classified into fall dormancy groups, numbered 1 to 10, which can be fitted into the plant hardiness zone map. Dormancy group 1 is very dormant and best suited for cold climates (such varieties would stop growing and go dormant over winter), and dormancy group 10 is very non-dormant and suited for very hot climates (such varieties would have high growth rates over a very long growing season and would have relatively high winter activity). For a comprehensive review of geographic adaptation of alfalfa, see Melton et al., Geographic Adaptation and Cultivar Selection 20: 595-620, In Alfalfa and Alfalfa Improvement, supra. Between 1900 and 1975 more than 160 cultivars were developed for production in North America. Most of the newer cultivars were selected for improved adaptation and multiple pest resistance. For a comprehensive review of the distribution, history and origin of alfalfa, see Michaud et al., World Distribution and Historical Development 2:25-91, In Alfalfa and Alfalfa Improvement, supra; and, Quiros et al., The Genus Medicago and the Origin of the Medicago sativa Complex 3:93-124, In Alfalfa and Alfalfa Improvement, supra.
The genus Medicago is widely distributed and comprises an array of diverse species that are either annual or perennial. The most recent taxonomic studies of the perennial species concluded that M. sativa is polymorphic. Lesins and Gillies (Taxonomy and cytogenetics of Medicago 353-386, In Alfalfa science and technology, C. H. Hanson (ed.), American Society of Agronomy, (1972)) defined the complex as M. sativa-falcata-glutinosa, and Gunn et al. (USDA Tech. Bull. No. 1574 (1978)) designated it as the M. sativa sensu lato complex.
M. sativa plants are autopolyploid organisms, or more specifically, autotetraploids. More specifically, M. sativa plants are polysomic polyploid organisms which display tetrasomic inheritance patterns.
Essentially all annual species are cleistogamous and are exclusively self-pollinated. Generally, the perennial species require tripping, as by insect visits to the floral structures, and will set seed from either self or cross-pollination. Crosses can be made among subspecies in the M. sativa complexes and between the cultivated tetraploids and wild diploids without special preparation of the parents. For a comprehensive review of the floral characteristics, plant culture, and methods of self-pollinating or hybridizing alfalfa, see D. K. Barnes, Alfalfa 9:177-187, In Hybridization of Crop Plants, Fehr et al. (ed.), American Society of Agronomy Inc. (1980).
Commercial alfalfa seed may be provided either in a synthetic variety or a hybrid variety. Commercial production of synthetic varieties may include a breeder seed production stage, a foundation seed production stage, a registered seed production stage and a certified seed production stage. Hybrid variety seed production may involve up to three stages including a breeder seed production stage, a foundation seed production stage and a certified seed production stage.
Efforts in developing healthy and productive alfalfa varieties often focus on breeding for disease and stress-resistant cultivars, for example, breeding for persistence, breeding for adaptation to specific environments, breeding for yield per se, and breeding for quality. Success has been attained in breeding for resistance to fungal, bacterial, insect, and nematode pests, including, but not limited to the development of varieties tolerant/resistant to bacterial wilt and common leaf spot (see, e.g., Elgin, Jr., et al., Breeding for Disease and Nematode Resistance 827-858, In Alfalfa and Alfalfa Improvement, supra) and to the spotted alfalfa aphid and alfalfa weevil (see, e.g., Sorensen et al., Breeding for Insect Resistance 859-902, In Alfalfa and Alfalfa Improvement, supra). Breeders have had less success in breeding for yield and quality per se (see, e.g., Hill et al., Breeding for Yield and Quality 26:809-825, In Alfalfa and Alfalfa Improvement, supra), although methods have been developed that help increase productivity and yield (U.S. Pat. No. 4,045,912). Historically, yield and productivity, quality and persistence are objectives of high concern to farmers.
In addition to conventional or classical plant breeding methodology, recombinant DNA technologies exist that allow alfalfa breeders to incorporate novel genes into alfalfa plants. Transgenic plants can now be produced by a variety of different transformation methods including, but not limited to, electroporation; microinjection; microprojectile bombardment, also known as particle acceleration or biolistic bombardment; viral-mediated transformation; and Agrobacterium-mediated transformation. Transgenic alfalfa plants have been produced by many of these methods including, but not limited to, Agrobacterium-mediated transformation (Wang et al., Australian Journal of Plant Physiology 23(3):265-270 (1996); Hoffman et al., Molecular Plant-Microbe Interactions 10(3):307-315 (1997); Trieu et al., Plant Cell Reports 16:6-11 (1996)) and particle acceleration (U.S. Pat. No. 5,324,646).
Various factors are important for the efficient transformation of crop plants. Of these variables, the ability to regenerate a complete plant from one or multiple cells of transformed explant tissue is one of the most important. Even when transformation has been achieved and is routine for a particular genotype of a crop species, it can still be difficult to transform any genotype of choice within a recalcitrant species. Numerous reports cite differences between genotypes within a species in the ability to regenerate in vitro (e.g. Diertert et al 1982; Christey and Earle, 1991). Such reports indicate that the regeneration processes are genetically controlled.
Alfalfa was one of the first major crop plants to regenerate somatic embryos from tissue culture (Saunders and Bingham, 1972). The ability of an alfalfa genotype to regenerate somatic embryos is believed to be genetically controlled by two genes. Bingham et al. (1975) were able to increase the frequency of plants which regenerate somatic embryos within a population of alfalfa through conventional breeding practices. Much of the published research in alfalfa transformation utilizes the alfalfa clone Regen SY as the source of explant material because of the suitability of this genotype for regeneration. Regen SY is a poorly adapted clone that lacks vegetative vigor, winterhardiness, resistance to many diseases and has a very poor phenotype compared to agronomically elite clones from conventional alfalfa breeding programs.
As demonstrated by this review, there is a real need for alfalfa plants that combine adaptation, productivity, winterhardiness, and disease resistance with high levels of somatic embryogenesis. The present invention provides alfalfa plants selected for improved somatic embryogenesis. The alfalfa plants provided by this invention combine adaptation, productivity, winterhardiness, and disease resistance with high levels of somatic embryogenesis.